For more than forty years, thymidylate metabolism has been an important biochemical target for widely utilized anti-cancer agents. Inhibitors of this pathway such as the fluoropyrimidines and antifolates induce a severe depletion of TTP pools resulting in nucleotide pool imbalance and cell killing through a process termed "thymineless death." Investigation of the underlying mechanisms of this process suggest that aberrant uracil-DNA metabolism may be an important mediator of DNA damage and cell killing. The broad, long-term objectives of this proposal are: 1) to elucidate the role of aberrant dUTP metabolism as a molecular mechanism of cell killing induced by chemotherapeutic agents that target thymidylate biosynthesis and; 2) to better understand the role of key enzymes involved in dUTP metabolism in modulating chemosensitivity. In this study, the applicants propose a mechanistic analysis of thymineless death using human colon cancer cell lines that over express the enzyme deoxyuridine triphosphate nucleotide hydrolase (dUTPase). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and PPi, thereby eliminating dUTP from the DNA biosynthetic pathway. The applicants hypothesize that dUTPase over-expression counters the cytotoxic effect of FUdR treatment by limiting the expansion of dUTP pools. Although there is significant evidence suggesting that uracil-DNA metabolism may be a critical factor in mediating cytotoxicity, there have been no biochemical studies performed to clarify the role of human dUTPase isoform expression in modulating chemosensitivity. The proposed studies are designed to correlate key mechanistic hallmarks of uracil-DNA mediated cytotoxicity with cell death induced by fluorodeoxyuridine. Specific Aim 1 investigates the role of dUTPase isoform over-expression as a mechanism of resistance to FUdR-induced cytotoxicity. Specific Aim 2 investigates the correlation between biochemical endpoints of aberrant uracil-DNA metabolism and chemosensitivity to FUdR. Specific Aim 3 investigates the significance of dUTPase isoform expression in predicting patient response to fluoropyrimidine-based chemotherapy and overall survival in metastatic colon cancer. A better understanding of the role of aberrant uracil-DNA metabolism in mediating thymineless death should not only enhance our knowledge of the molecular mechanism of drug action of these important chemotherapeutics, but also provide insight into novel and improved treatment strategies.